The purpose is to perform carefully controlled vaccine-based trials to determine the optimal therapeutic approach by which tumor reactive T lymphocytes can be safely and efficiently generated for treatment of advanced melanoma patients, coupled with alternative methods of immunological monitoring that move beyond changes in the blood to detailed cellular and molecular tissue-based analyses. Since melanoma will be diagnosed in 43,000 new patients annually, and 7,300 deaths result from melanoma, new treatment approaches are indicated because melanoma generally progresses despite conventional chemotherapy and radiation. A promising approach for melanoma involves immunotherapy in which the patient's immuno system is stimulated to generate effective anti-tumor responses. The primary antigen presenting cell used in these trials are dendritic cells (DCs) pulsed with autologous tumor-cell lysate containing T-cell stimulatory tumor cell-derived antigens. Incentives for this immunotherapy approach include the capacity of appropriately selected DCs to stimulate both CD4+ as well as CD8+ effector T cells. Our therapeutic strategy is designed to by-pass several known methods by which melanoma cells normally thwart the immune system. Thus, we will expand both immature and mature DCs ex-vivo (using CD40L) apart from the systemic immunosuppressive factors produced by melanoma cells, and create lysates from autologous tumor cells. Studies are designed to identify immunological in-vivo activity in blood, and at various tissue sites for mature versus immature DCs, and the preferred method of vaccination, to generate the highest number of cytotoxic T cells and positive clinical responses. Since many Stage IV patients will not respond to treatment with complete remissions, focus will be directed at methods to monitor and explore the mechanistic basis by which tumor cells escape the activated immune response following vaccination. Specific protocols include an initial randomized Phase I study of 24 patients to compare immature DCs versus mature DCs loaded with tumor lysate-containing antigens injected intradermally, followed by a randomized Phase II clinical trial of 40 patients using the best DC (immature vs. mature) and comparing intradermal versus intranodal routes of delivery. Immunological monitoring of patients will include a comparison of traditional blood-derived markers versus assays performed on cells with serial gene profiling of samples derived from sequential fine needle aspirates of metastatic melanoma lesions before and after vaccination, as well as normal lymph nodes and the sentinel node draining from vaccination sites. The vaccination site itself will also be examined in the phase I trial comparing the local tissue response to mature versus immature DCs. Taken together the guiding tenant of this grant is that we can best mimic the physiological protective immune pathway by carefully isolating specific types of DCs and exposing them ex-vivo to appropriate tumor antigens, followed by re-infusing them in a manner that takes advantage of the immunological potency of DCs, with concommitant avoidance or by-passing local and systemic immunosuppressive or immune-escape mechanisms created by melanoma cells. In this way greater understanding and better treatment protocols and immunological monitoring strategies will be developed for patients undergoing vaccination to eliminate metastatic melanoma.